Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro.

Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases.

In vitro degradation and cell viability assessment of Zn-3Mg alloy for biodegradable bone implants.

This article reports the in vitro degradation and cytotoxicity assessment of Zn-3Mg alloy developed for biodegradable bone implants. The alloy was prepared using casting, and its microstructure was composed of Mg2Zn11 intermetallic phase distributed within a Zn-rich matrix. The degradation assessment was done using potentiodynamic polarization and electrochemical impedance spectrometry. The cell viability and the function of normal human osteoblast cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and alkaline phosphatase extracellular enzyme activity assays. The results showed that the degradation rate of the alloy was slower than those of pure Zn and pure Mg due to the formation of a high polarization resistance oxide film. The alloy was cytocompatible with the normal human osteoblast cells at low concentrations (<0.5 mg/mL), and its alkaline phosphatase activity was superior to pure Mg. This assessment suggests that Zn-3Mg alloy has the potential to be developed as a material for biodegradable bone implants, but the toxicity limit must be carefully observed.

Cytotoxicity evaluation of biodegradable Zn-3Mg alloy toward normal human osteoblast cells.

The recent proposal of using Zn-based alloys for biodegradable implants was not supported with sufficient toxicity data. This work, for the first time, presents a thorough cytotoxicity evaluation of Zn-3Mg alloy for biodegradable bone implants. Normal human osteoblast cells were exposed to the alloy's extract and three main cell-material interaction parameters: cell health, functionality and inflammatory response, were evaluated. Results showed that at the concentration of 0.75mg/ml alloy extract, cell viability was reduced by ~50% through an induction of apoptosis at day 1; however, cells were able to recover at days 3 and 7. Cytoskeletal changes were observed but without any significant DNA damage. The downregulation of alkaline phosphatase protein levels did not significantly affect the mineralization process of the cells. Significant differences of cyclooxygenase-2 and prostaglandin E2 inflammatory biomarkers were noticed, but not interleukin 1-beta, indicating that the cells underwent a healing process after exposure to the alloy. Detailed analysis on the cell-material interaction is further discussed in this paper.

Tocotrienol preserves ovarian function in cyclophosphamide therapy.

INTRODUCTION: Cyclophosphamide (CPA) chemotherapy leads to ovarian failure and infertility. Tocotrienol (T3) is an antioxidant and anti-inflammatory agent. The role of T3 in ovarian protection throughout chemotherapy remains unclear. AIM: To investigate the role of T3 in the preservation of female fertility in CPA treatment. METHOD: Sixty female mice were divided into five treatment groups, namely, normal saline, corn oil only, T3 only, CPA and CPA + T3. The treatment was given for 30 days, followed by administration of gonadotrophin to induce ovulation. After killing, both ovaries were collected and examined histologically. RESULTS: There was significant reduction in ovarian size in the CPA group compared with the normal group (CPA versus normal, mean area ± SD; 0.118 ± 0.018 vs. 0.423 ± 0.024 cm(2); p ≤ 0.005), whilst concurrent administration of T3 with CPA leads to conservation of ovarian size (CPA + T3 vs. CPA, mean area ± SD; 0.285 ± 0.032 vs. 0.118 ± 0.018 cm(2); p ≤ 0.005). Ovaries in CPA group showed abnormal folliculogenesis with accompanied reduced ovulation rate, follicular oedema, increased vascularity and inflammatory cell infiltration. These changes were reversed by concurrent T3 administration. CONCLUSION: Co-administration of T3 with CPA confers protection of ovarian morphology and function in vivo. These findings contribute to the further elucidation of CPA effect on ovary and suggest the potential of T3 use in preserving fertility in chemotherapy.

Short-term moderate hypothermia stimulates alkaline phosphatase activity and osteocalcin expression in osteoblasts by upregulating Runx2 and osterix in vitro.

Exposure of Normal Human Osteoblast cells (NHOst) to a period of hypothermia may interrupt their cellular functions, lead to changes in bone matrix and disrupt the balance between bone formation and resorption, resulting in bone loss or delayed fracture healing. To investigate this possibility, we exposed NHOst cells to moderate (35 °C) and severe (27 °C) hypothermia for 1, 12, 24 and 72 h. The effects of hypothermia with respect to cell cytoskeleton organization, metabolic activity and the expression of cold shock chaperone proteins, osteoblast transcription factors and functional markers, were examined. Our findings showed that prolonged moderate hypothermia retained the polymerization of the cytoskeletal components. NHOst cell metabolism was affected differently according to hypothermia severity. The osteoblast transcription factors Runx2 and osterix were necessary for the transcription and translation of bone matrix proteins, where alkaline phosphatase (Alp) activity and osteocalcin (OCN) bone protein were over expressed under hypothermic conditions. Consequently, bone mineralization was stimulated after exposure to moderate hypothermia for 1 week, indicating bone function was not impaired. The cold shock chaperone protein Rbm3 was significantly upregulated (p<0.001) during the cellular stress adaption under hypothermic conditions. We suggest that Rbm3 has a dual function: one as a chaperone protein that stabilizes mRNA transcripts and a second one in enhancing the transcription of Alp and Ocn genes. Our studies demonstrated that hypothermia permitted the in vitro maturation of NHOst cells probably through an osterix-dependent pathway. For that reason, we suggest that moderate hypothermia can be clinically applied to counteract heat production at the fracture site that delays fracture healing.

Morphological and phenotypical characteristics of human osteoblasts after short-term space mission.

Morphological and phenotypical signs of cultured readaptation osteoblasts were studied after a short-term space mission. The ultrastructure and phenotype of human osteoblasts after Soyuz TMA-11 space flight (2007) were evaluated by scanning electron microscopy, laser confocal microscopy, and ELISA. The morphofunctional changes in cell cultures persisted after 12 passages. Osteoblasts retained the drastic changes in their shape and size, contour deformation, disorganization of the microtubular network, redistribution of organelles and specialized structures of the plasmalemma in comparison with the ground control cells. On the other hand, the expression of osteoprotegerin and osteocalcin (bone metabolism markers) increased; the expression of bone resorption markers ICAM-1 and IL-6 also increased, while the expression of VCAM-1 decreased. Hence, space flight led to the development of persistent shifts in cultured osteoblasts indicating injuries to the cytoskeleton and the phenotype changes, indicating modulation of bone metabolism biomarkers.

Alteration of cell cytoskeleton and functions of cell recovery of normal human osteoblast cells caused by factors associated with real space flight.

Experiments involving short-term space flight have shown an adverse effect on the physiology, morphology and functions of cells investigated. The causes for this effect on cells are: microgravity, temperature fluctuations, mechanical stress, hypergravity, nutrient restriction and others. However, the extent to which these adverse effects can be repaired by short-term space flown cells when recultured in conditions of normal gravity remains unclear. Therefore this study aimed to investigate the effect of short-term spaceflight on cytoskeleton distribution and recovery of cell functions of normal human osteoblast cells. The ultrastructure was evaluated using ESEM. Fluorescent staining was done using Hoechst, Mito Tracker CMXRos and Tubulin Tracker Green for cytoskeleton. Gene expression of cell functions was quantified using qPCR. As a result, recovered cells did not show any apoptotic markers when compared with control. Tubulin volume density (p<0.001) was decreased significantly when compared to control, while mitochondria volume density was insignificantly elevated. Gene expression for IL-6 (p<0.05) and sVCAM-1 (p<0.001) was significantly decreased while alkaline phosphatase (p<0.001), osteocalcin and sICAM (p<0.05) were significantly increased in the recovered cells compared to the control ones. The changes in gene and protein expression of collagen 1A, osteonectin, osteoprotegerin and beta-actin, caused by short-term spaceflight, were statistically not significant. These data indicate that short term space flight causes morphological changes in osteoblast cells which are consistent with hypertrophy, reduced cell differentiation and increased release of monocyte attracting proteins. The long-term effect of these changes on bone density and remodeling requires more detailed studies.

Interleukin-6 and intercellular cell adhesion molecule-1 expression remains elevated in revived live endothelial cells following spaceflight.

The effects of spaceflight on cardiovascular health are not necessarily seen immediately after astronauts have returned but can be delayed. It is important to investigate the long term effects of spaceflight on protein and gene expression of inflammation and endothelial activation as a predictor for the development of atherosclerosis and potential cardiovascular problems. The objectives of this study were to investigate the (a) protein and gene expression of inflammation and endothelial activation, (b) expression of nuclear factor kappa B (NFκB), signal transducer and activator of transcription-3 (STAT-3) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVEC) 3 months post-space flight travel compared to ground controls. HUVEC cultured on microcarriers in fluid processing apparatus were flown to the International Space Station (ISS) by the Soyuz TMA-11 rocket. After landing, the cells were detached from microcarriers and recultured in T-25 cm(2) culture flasks (Revived HUVEC). Soluble protein expression of IL-6, TNF-α, ICAM-1, VCAM-1 and e-selectin were measured by ELISA. Gene expression of these markers and in addition NFκB, STAT-3 and eNOS were measured. Spaceflight induced IL-6 and ICAM-1 remain elevated even after 3 months post spaceflight travel and this is mediated via STAT-3 pathway. The downregulation of eNOS expression in revived HUVEC cells suggests a reduced protection of the cells and the surrounding vessels against future insults that may lead to atherosclerosis. It would be crucial to explore preventive measures, in relation to atherosclerosis and its related complications.

Effects of space mission factors on the morphology and function of endothelial cells.

The structure and functions of endothelial cells after space mission were studied by electron and laser confocal microscopy, image analysis, and MTT test. The endothelial cells changed significantly (proliferative activity, size, contours, shape, distribution of mitochondria and microtubules) in comparison with controls on the Earth. These changes indicated injuries in the cytoskeleton and impairment of the barrier function of the cells, which presumably contributed to the development of endothelial dysfunction.

Real space flight travel is associated with ultrastructural changes, cytoskeletal disruption and premature senescence of HUVEC.

Microgravity, hypergravity, vibration, ionizing radiation and temperature fluctuations are major factors of outer space flight affecting human organs and tissues. There are several reports on the effect of space flight on different human cell types of mesenchymal origin while information regarding changes to vascular endothelial cells is scarce. Ultrastructural and cytophysiological features of macrovascular endothelial cells in outer space flight and their persistence during subsequent culturing were demonstrated in the present investigation. At the end of the space flight, endothelial cells displayed profound changes indicating cytoskeletal lesions and increased cell membrane permeability. Readapted cells of subsequent passages exhibited persisting cytoskeletal changes, decreased metabolism and cell growth indicating cellular senescence.

Native skeletal muscle dihydropyridine receptor exists as a supramolecular triad complex.

One of the central elements of excitation-contraction coupling, the voltage-sensing dihydropyridine receptor, is believed to exist as a high-molecular-mass complex in the triad junction. Although freeze-fracture electron microscopical analysis suggests a tetrad complex, no direct biochemical evidence exists demonstrating the actual size of the native membrane complex. Using a combination of various two-dimensional gel electrophoresis techniques, we show here that the principal alpha1-subunit of the dihydropyridine receptor and its auxiliary alpha2-subunit form a triad complex of approximately 2800 kDa under native conditions. Established Ca2+-ATPase tetramers and calsequestrin monomers were employed for the internal standardization of the gel systems used. Thus, the large voltage-sensing complex appears to be tightly associated, since it does not disintegrate during subcellular fractionation and native electrophoresis procedures. Our findings support the cell biological hypothesis that native dihydropyridine receptor units form a tetrad structure within the transverse tubules.

Low-frequency stimulation of fast muscle affects the abundance of Ca(2+)-ATPase but not its oligomeric status.

After chronic, low-frequency stimulation, a rapid decline in Ca(2+) pump activity is observed during the early stages of skeletal muscle transformation. However, this variation in enzymatic activity does not coincide with a drastic reduction in the amount of sarcoplasmic reticulum Ca(2+)-ATPases. To investigate whether changes in subunit interactions within Ca(2+) pump complexes contribute to this phenomena, we performed a chemical cross-linking analysis of 4 days continuously, and 4 days discontinuously, electrostimulated fast muscle fibers. The abundance of the slow and fast Ca(2+)-ATPase isoforms sarco(endo)plasmic reticulum Ca(2+)- ATPase types 1 and 2 was affected during the fast-to-slow transition process, demonstrating that, even after short-term stimulation, distinct changes in the isoform expression pattern of muscle proteins occur. However, the oligomeric status of both ion pump species did not change. Hence, chemical modifications of critical enzyme domains must be responsible for the rapid stimulation-induced activity changes, not variations in protein-protein interactions within Ca(2+)-ATPase units. Oligomerization appears to be of central importance to the proper physiological functioning of the Ca(2+)-ATPase and does not undergo changes during skeletal muscle conditioning.

The role of ion-regulatory membrane proteins of excitation-contraction coupling and relaxation in inherited muscle diseases.

The excitation-contraction-relaxation cycle of skeletal muscle fibres depends on the finely tuned interplay between the voltage-sensing dihydropyridine receptor, the junctional ryanodine receptor Ca2+-release channel and the sarcoplasmic reticulum Ca2+-ATPase. Inherited diseases of excitation-contraction coupling and muscle relaxation such as malignant hyperthermia, central core disease, hypokalemic periodic paralysis or Brody disease are caused by mutations in these Ca2+-regulatory elements. Over twenty different mutations in the Ca2+-release channel are associated with susceptibility to the pharmacogenetic disorder malignant hyperthermia. Other mutations in the ryanodine receptor trigger central core disease. Primary abnormalities in the alpha-1 subunit of the dihydropyridine receptor underlie the molecular pathogenesis of both hypokalemic periodic paralysis and certain forms of malignant hyperthermia. Some cases of the muscle relaxation disorder named Brody disease were demonstrated to be based on primary abnormalities in the Ca2+-ATPase. Since a variety of other sarcoplasmic reticulum proteins modulate the activity of the voltage sensor, Ca2+-release channel and ion-binding proteins, mutations in these Ca2+-regulatory muscle components might be the underlying cause for novel, not yet fully characterized, genetic muscle disorders. The cell biological analysis of knock-out mice has been helpful in evaluating the biomedical consequences of defects in ion-regulatory muscle proteins.

Comparative analysis of the isoform expression pattern of Ca(2+)-regulatory membrane proteins in fast-twitch, slow-twitch, cardiac, neonatal and chronic low-frequency stimulated muscle fibers.

Although all muscle cells generate contractile forces by means of organized filament systems, isoform expression patterns of contractile and regulatory proteins in heart are not identical compared to developing, conditioned or mature skeletal muscles. In order to determine biochemical parameters that may reflect functional variations in the Ca(2+)-regulatory membrane systems of different muscle types, we performed a comparative immunoblot analysis of key membrane proteins involved in ion homeostasis. Cardiac isoforms of the alpha(1)-dihydropyridine receptor, Ca(2+)-ATPase and calsequestrin are also present in skeletal muscle and are up-regulated in chronic low-frequency stimulated fast muscle. In contrast, the cardiac RyR2 isoform of the Ca(2+)-release channel was not found in slow muscle but was detectable in neonatal skeletal muscle. Up-regulation of RyR2 in conditioned muscle was probably due to degeneration-regeneration processes. Fiber type-specific differences were also detected in the abundance of auxiliary subunits of the dihydropyridine receptor, the ryanodine receptor and the Ca(2+)-ATPase, as well as triad markers and various Ca(2+)-binding and ion-regulatory proteins. Hence, the variation in innervation of different types of muscle appears to have a profound influence on the levels and pattern of isoform expression of Ca(2+)-regulatory membrane proteins reflecting differences in the regulation of Ca(2+)-homeostasis. However, independent of the muscle cell type, key Ca(2+)-regulatory proteins exist as oligomeric complexes under native conditions.

Effect of halothane on the oligomerization of the sarcoplasmic reticulum Ca(2+)-ATPase.

The exact molecular mechanism of inhalational anesthetics remains obscure. Since the enzyme activity of the sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle fibres is modified by halothane and because protein-protein interactions play an important role in the regulation of Ca(2+)-regulatory proteins, we investigated the effect of this volatile drug on the oligomerization of the fast-twitch Ca(2+)-ATPase. Using electrophoretic separation following incubation with halothane, increases in relative molecular mass were determined by immunoblotting with a monoclonal antibody to the SERCA1 isoform of the Ca(2+)-ATPase. Distinct drug-induced decreases in electrophoretic mobility indicated oligomerization of the native Ca(2+)-pump by halothane, comparable to crosslinking-mediated formation of homo-tetramers. Determination of the effect of halothane on enzyme activity suggested that halothane-mediated protein aggregation triggers a partial inhibition of Ca(2+)-pump units. Thus, halothane appears to exert its action via specific peptide binding sites and not indirectly by lipid perturbation. These findings support the protein theory of anesthetic action.

The 90-kDa junctional sarcoplasmic reticulum protein forms an integral part of a supramolecular triad complex in skeletal muscle.

Although it is well established that voltage-sensing of the alpha(1)-dihydropyridine receptor triggers Ca(2+)-release via the ryanodine receptor during excitation-contraction coupling in skeletal muscle fibers, it remains to be determined which junctional components are responsible for the assembly, maintenance, and stabilization of triads. Here, we analyzed the expression pattern and neighborhood relationship of a novel 90-kDa sarcoplasmic reticulum protein. This protein is highly enriched in the triad fraction and is predominantly expressed in fast-twitching muscle fibers. Chronic low-frequency electro-stimulation induced a drastic decrease in the relative abundance of this protein. Chemical crosslinking showed a potential overlap between the 90-kDa junctional face membrane protein and the ryanodine receptor Ca(2+)-release channel, suggesting tight protein-protein interactions between these two triad components. Hence, Ca(2+)-regulatory muscle proteins have a strong tendency to oligomerize and the triad region of skeletal muscle fibers forms supramolecular membrane complexes involved in the regulation of Ca(2+)-homeostasis and signal transduction.

Self-aggregation of triadin in the sarcoplasmic reticulum of rabbit skeletal muscle.

The 95 kDa transmembrane glycoprotein triadin is believed to be an essential component of excitation-contraction coupling in the junctional sarcoplasmic reticulum of skeletal muscle fibers. It is debatable whether triadin mediates intraluminal interactions between calsequestrin and the ryanodine receptor exclusively or whether this junctional protein provides also a cytoplasmic linkage between the Ca2+-release channel and the dihydropyridine receptor. Here, we could show that native triadin exists as disulfide-linked homo-polymers of above 3000 kDa. Under non-reducing conditions, protein bands representing the alpha1-dihydropyridine receptor and calsequestrin did not show an immunodecorative overlap with the extremely high-molecular-mass triadin clusters. Following chemical crosslinking, the ryanodine receptor and triadin exhibited a similarly decreased electrophoretic mobility. However, immunoblotting of diagonal non-reducing/reducing two-dimensional gels clearly demonstrated a lack of overlap between the immunodecorated bands representing triadin, the alpha1-dihydropyridine receptor, the ryanodine receptor and calsequestrin. Thus, in native membranes triadin appears to form large self-aggregates primarily. Although triadin exists in a close neighborhood relationship to the Ca2+-release channel tetramers, it does not seem to be directly linked to the other main triad components implicated in the regulation of the excitation-contraction-relaxation cycle and Ca2+-homeostasis. This agrees with a proposed role of triadin in the maintenance of overall triad architecture.

Complex formation of skeletal muscle Ca2+-regulatory membrane proteins by halothane.

In skeletal muscle, halothane affects the functions of several Ca2+-regulatory membrane proteins involved in the excitation-contraction-relaxation cycle. To investigate the mechanism by which this volatile anesthetic interferes with Ca2+-homeostasis, we studied potential changes in protein-protein interactions by halothane. Using comparative immunoblotting of microsomal muscle proteins separated on native and denaturing gels, we show here that halothane induces oligomerization of the terminal cisternae Ca2+-binding protein calsequestrin, the junctional ryanodine receptor Ca2+-release channel and the transverse-tubular alpha1-dihydropyridine receptor. This agrees with previous reports on the modulation of Ca2+-release activity by halothane since interactions between the voltage-sensing alpha1-dihydropyridine receptor, the ryanodine receptor and the luminal Ca2+-reservoir might result in a rapid release of Ca2+-ions. Furthermore, this study supports the idea that specific protein sites are involved in the action of inhalational anesthetics and that halothane might trigger abnormal Ca2+-homeostasis in malignant hyperthermia via oligomerization of the mutated ryanodine receptor.

Excitation-contraction-relaxation cycle: role of Ca2+-regulatory membrane proteins in normal, stimulated and pathological skeletal muscle (review).

Extremely large protein complexes involved in the Ca2+-regulatory system of the excitation-contraction-relaxation cycle have been identified in skeletal muscle, i.e. clusters of the Ca2+-binding protein calsequestrin, apparent tetramers of Ca2+-ATPase pump units and complexes between the transverse-tubular alpha1-dihydropyridine receptor and ryanodine receptor Ca2+-release channel tetramers of the sarcoplasmic reticulum. While receptor interactions appear to be crucial for signal transduction during excitation-contraction coupling, avoidance of passive disintegration of junctional complexes and stabilization of receptor interactions may be mediated by disulfide-bonded clusters of triadin. Oligomerization of Ca2+-release, Ca2+-sequestration and Ca2+-uptake complexes appear to be an intrinsic property of these muscle membrane proteins. During chronic low-frequency stimulation, the expression of triad receptors is decreased while conditioning has only a marginal effect on Ca2+-binding proteins. In contrast, muscle stimulation induces a switch from the fast-twitch Ca2+-ATPase to its slow-twitch/cardiac isoform. These alterations in Ca2+-handling might reflect early functional adaptations to electrical stimulation. Studying Ca2+-homeostasis in transformed muscles is important regarding the evaluation of new clinical applications such as dynamic cardiomyoplasty. Studies of Ca2+-handling in skeletal muscle fibers have not only increased our understanding of muscle regulation, but have given important insights into the molecular pathogenesis of malignant hyperthermia, hypokalemic periodic paralysis and Brody disease.

Oligomerisation of Ca2+-regulatory membrane components involved in the excitation-contraction-relaxation cycle during postnatal development of rabbit skeletal muscle.

The skeletal muscle excitation-contraction-relaxation cycle matures during the first weeks after birth and protein-protein interactions are believed to be essential for proper Ca2+ regulation. We therefore studied potential changes in the oligomerisation of key components of the Ca2+-regulatory membrane system during postnatal myogenesis. In contrast to a decrease in calreticulin, the Ca2+-binding proteins calsequestrin and sarcalumenin increased in abundance in microsomes isolated from muscle between postnatal days 1 and 41. While the expression of the fast Ca2+-ATPase increased, its slow-twitch isoform decreased. The junctional component triadin, the 53 kDa sarcoplasmic reticulum glycoprotein, as well as the dihydropyridine receptor increased in abundance, while no major changes in the expression of the ryanodine receptor were observed. Crosslinking analysis revealed that the fast Ca2+-ATPase, alpha1-dihydropyridine receptor and calsequestrin exhibit a more pronounced tendency to oligomerise in adult muscle fibres as compared to early postnatal stages. Interestingly, adult calsequestrin exists not only as a 63 kDa form but also as stable molecular species of higher molecular mass. These findings imply that during postnatal development, protein-protein interactions within the Ca2+-regulatory membrane system become more complex and oligomerisation appears to be an essential prerequisite for the proper physiological functioning of key membrane proteins in matured skeletal muscle fibres.